When stable isotopes are substituted for atoms undergoing a chemical reaction, it changes the rates and equilibria of the reactions in which the substituted atom is involved. By analyzing these isotope effects (lEs) for enzyme-catalyzed reactions one can elucidate details of enzyme mechanism and provide key information about active site interactions. This information is essential for understanding how the enzymes that are essential for human life function. Importantly, this information can be used to design specific inhibitors of enzyme function that can be used to stop the growth of cancer and viruses. However, due to technical limitations IE analysis is not widely applied to enzymes acting on DMA and RNA. These enzymes are of intense interest because they are fundamental to biology and are important targets for therapeutics, yet gaining direct information on their mechanism has been difficult. Defining the mechanism of nucleic acid phosphoryl transfer enzymes by competitive IE analyses faces two substantial challenges- first, sufficient amounts of DNA or RNA with site-specific isotopic enrichment must be synthesized, and second, the isotopic composition of the product must be precisely determined by mass spectrometry (MS). Our recent results on nucleophile clearly demonstrate the feasibility of using a new analytical technique, whole molecule electrospray ionization MS (ESI-MS), for measuring lEs on reactions of RNA oligonucleotides. Additionally, we have observed that advances in tandem quadropol / time of flight (Q/TOF) ESI-MS now permit analysis of very small amounts of material with increased precision necessary for detailed IE analysis of enzyme mechanism. In this project we will develop Q/TOF ESI-MS together with standard nucleoside modification chemistry using inexpensive and commercially available reagents to permit the lEs on each of the oxygens of a single, specific scissile phosphate in an oligonucleotide substrate. Such information will set the stage for the future design of active site based inhibitors as anti-proliferative (anri- cancer) or anti viral drugs. [unreadable] [unreadable] [unreadable]